This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The FRETR metric was developed to measure FRET between CFP and YFP. It replaces other metrics which generally are nonlinear and are insensitive to low values. FRETR is relative, empirical measure of FRET. It expresses the fold increase of fluorescence from FRET over a baseline derived from the inherent fluorescent properties of CFP and YFP. In our study on the organization of the spindle pole body (SPB), FRETR values were divided into 5 broad categories: none, lowest, low, moderate and high. Our model of the SPB and cryo-EM images of the SBP put limits on the possible distances between CFP and YFP for each class. This project will further explore the relationship between FRETR values and the distance between fluorophores.